Extended Data Fig. 8: Fast and slow tracking mode of single-molecule tracking.
a, Fast tracking with high laser power and short frame rate (10 ms/frame) reveals diffusive dynamics of free (red) and bound (blue) molecules. b, Single-step disappearance of chromatin-bound RNAPII in fast tracking. c, Slow tracking with low laser power and long frame rate (250 ms/frame) reveals residence time of bound molecules (blue). d, Single-step disappearance of chromatin-bound RNAPII in slow tracking. b-d, Here we term ‘disappearance’ as we cannot definitively differentiate between the molecule diffusing out of the focal plane or photobleaching within the living cell. e, CDF of bound displacement of RNAPII, H2B, H3 and H2A.Z to determine the rmax for trajectory linking in slow tracking100. f, In G1 cells, nucleosomal H2B (representing the stable binding population) was hypothesized to bind for a sufficiently long period, such that during image acquisition, their dissociation was barely observed. Nonetheless, the observed residence times for H2B typically fall within 30 seconds, due to factors such as photobleaching, dye blinking, chromatin/nuclear movements, and focus drift. g, Survival probability of Halo-H2B observed residence times in CTD26 (WT) and CTD9 strains (n: number of trajectories; mean value ± s.d.). h, Decay constant of stably bound Halo-H2B (ksb) in CTD26 (WT) and CTD9 strains (n = 10,000 resamplings; mean value ± s.d.; two-tailed unpaired t-test, ns P = 0.339598). Source numerical data are available in source data.
